Cytodiagnosis is the diagnosis of disease through the

microscopic examination of cells (of human, animal or

plant origin) collected by various means.
In the case of human cytodiagnosis, there are two areas of
cytology gynecological and non-gynecological in which
specimen material is examined for the presence of
malignant and premalignant cells, which, through certain
procedures, may be classified as:
Normal

Inflammatory
Suspect / Uncertain
Malignant

WHAT IS PAP TEST?

Pap test is a method of examining with a microscope a sample of
superficial cells that line the inner wall of the uterine cervix to detect
any abnormal cell for early diagnosis of uterine cancer.

It was developed in 1928 by the Greek doctor George Nicholas

Papanicolaou (1883-1962) at the Cornell Medical College of New
York. He also developed the particular polychrome staining reaction
designed to demonstrate variations of cellular maturity and
metabolic activity.
The name Pap test" derives from the first letters of his surname.

The Papanicolaou stain is also used for nongynecological (clinical) material. For instance,
specimens of sputum or urine, containing squamous
epithelial or similar cells, demonstrate
excellent results when stained according to the
Papanicolaou technique.

Papanicolaou stain

Papanicolaou stain
The three main advantages of this staining procedure are:
(1) Good definition of nuclear detail.
(2) Cytoplasmic transparency.
(3) Indication of cellular differentiation of squamous
epithelium.
It is a polychrome staining method which depends on
degree of cell maturity and cellular metabolic activity.
Cytoplasmic transparency is a function of high ethanol
concentration of the stain. This is important in order to
view multilayered cell agregates.

PRINCIPLES OF PAP STAIN

HAEMATOXILYN (violet): a basic stain with chemical
affinity for acid substances (i.e. nuclei of cells, filled with
DNA).
EA50 (light blue): an acid stain that reacts with the
cytoplasm of less mature squamous (exocervical) cell
(basal, parabasal and intermediate cell) and with
glandular (endocervical) cell.

OG6 (orange): an acid stain that reacts with superficial

squamous cell, filled with keratin.

 The haematoxylin nuclear stain demonstrates

chromatinic patterns of normal and abnormal cells.
The counterstains, Orange-G and E.A. (eosin-azure) have
a high alcoholic concentration which provides cytoplasmic
transparency. This enables clear visualization through
areas of overlapping cells, mucus and debris.
There are four main steps in the staining procedure:
(1) Fixation.

(2) Nuclear staining.

(3) Cytoplasmic staining.
(4) Clearing.

Staining steps
1) Ethanol 95 (Fixation)
2) Distilled water
3) Harris Hematoxylin
4) Tap water
5) Ethanol 95
6) OG 6
7) Ethanol 95
8) EA 50
9) Ethanol 95
10) Absolute Ethanol
11) Xilene or Bio Clear

2 minutes
2 minutes
1 minute
5 minutes
15 seconds
2 minutes
15 seconds (twice)
5 minutes
15 seconds
30 seconds (twice)
2 minutes (twice)

Fixation
Specimens must be fixed immediately after being taken and while
still moist!!!!
To prevent drying out and shrinking of cells

To maintain specimens structural features

To permit clear staining and differentiation
If specimens are fixed too late, so-called artifacts can be found in
Papanicolaou-stained smears on single cells or cell clusters.
The classic method of fixing is to immerse the microscope slides in
96% ethanol for 30 min.
A more efficient and quicker way is to fix them with a spray fixative.
Spray fixatives are aqueous-alcoholic solutions containing
polyethylene glycol (PEG, Carbowax), and are suitable for all types of
cytological material due to be stained by the Papanicolaou method.

Air drying artifacts

Air drying is a physiochemical process where there is more or less
complete loss of water from the cells (especially from the nuclei)
connected with structural alterations of the cell, as spreading of
the nucleus or reduction of the staining reaction after application
of the PAP stain.

Alterations caused by airdrying:

1. Spreading of cells on the slide surface with a change of
nuclear area. 3D cell nuclei become flat,
2. Condensation of chromatin. This cannot be fully restored after
reimmersion in water,
3. Favouring/preventing staining reactions.

Staining steps
1) Ethanol 95 (Fixation)
2) Distilled water
3) Harris Hematoxylin
4) Tap water
5) Ethanol 95
6) OG 6
7) Ethanol 95
8) EA 50
9) Ethanol 95
10) Absolute Ethanol
11) Xilene or Bio Clear

2 minutes
2 minutes
1 minute
5 minutes
15 seconds
2 minutes
15 seconds (twice)
5 minutes
15 seconds
30 seconds (twice)
2 minutes (twice)

 From fixative (95% alcohol) the cells are hydrated through

a graded series of alcohols to water preparatory to
haematoxylin immersion (the haematoxylin is an aqueous
solution).

The cells are then dehydrated prior to immersion in the

alcohol based cytoplasmic counterstains.
Grading the alcoholic solutions in a stepwise manner is
thought to minimise cellular distortion and reduce cell loss
from the glass slide, due to convection currents in the
solutions.

Hematoxylin
The haematoxylin nuclear
stain is a natural stain which
has been used for over 100
years in histology.
It has affinity for
chromatin, attaching to
sulphate groups on the
DNA. molecule.
Rinse under tap water to
remove excess dye

Staining steps
1) Ethanol 95 (Fixation)
2) Distilled water
3) Harris Hematoxylin
4) Tap water
5) Ethanol 95
6) OG 6
7) Ethanol 95
8) EA 50
9) Ethanol 95
10) Absolute Ethanol
11) Xilene or Bio Clear

2 minutes
2 minutes
1 minute
5 minutes
15 seconds
2 minutes
15 seconds (twice)
5 minutes
15 seconds
30 seconds (twice)
2 minutes (twice)

Orange G
A monochromatic stain which colours keratin
a brilliant orange.
The effects of Orange G are only evident in
smear when keratinised cells are present.
However it is likely that it enhances red blood
cell staining.

Staining steps
1) Ethanol 95 (Fixation)
2) Distilled water
3) Harris Hematoxylin
4) Tap water
5) Ethanol 95
6) OG 6
7) Ethanol 95
8) EA 50
9) Ethanol 95
10) Absolute Ethanol
11) Xilene or Bio Clear

2 minutes
2 minutes
1 minute
5 minutes
15 seconds
2 minutes
15 seconds (twice)
5 minutes
15 seconds
30 seconds (twice)
2 minutes (twice)

EA-50 (Eosin Azure)

a polychromatic mixture of:
1.Eosin G
2.Light Green SF
3.Bismarck Brown
Various EA modifications are known. They
differ simply through the various concentrations
of the individual dyes.

Staining solutions commonly used in cytology

are EA 31 and EA 50, while EA 65 is preferred for
mucous material such as sputum, bronchial
secretions and other non-gynecological material.
Bismarck Brown reportedly does not have a
staining effect but rather contributes to
stabilizing the staining solution.

 The two dyes Eosin G and

Light Green SF compete for
the same target structures
and cause the cells to be
differently stained at various
cyclic stages.
Mature squamous epithelial
cells, nucleoli and ciliae, for
instance, have a stronger
affinity for Eosin G, while
parabasal and intermediate
cells appear green, blue-green
or blue after being stained
with Light Green SF.

Omission of Orange G did

not affect the accuracy of
diagnosis (since keratin
and red blood cell are also
stained by eosin).

Staining steps
1) Ethanol 95 (Fixation)
2) Distilled water
3) Harris Hematoxylin
4) Tap water
5) Ethanol 95
6) OG 6
7) Ethanol 95
8) EA 50
9) Ethanol 95
10) Absolute Ethanol
11) Xilene or Bio Clear

2 minutes
2 minutes
1 minute
5 minutes
15 seconds
2 minutes
15 seconds (twice)
5 minutes
15 seconds
30 seconds (twice)
2 minutes (twice)

Clearing
Clearing in xylol results in cellular
transparency and precedes mounting.
Xylol is the commonest clearing agent and is
miscible with alcohol (absolute only).
Xylol is colorless, chemically non-reactive and
has almost the same refractive index as glass
which is important to give the best possible
transparency of the image.
The presence of water in xylol causes
cloudiness due to water droplets. Water and
xylol are immiscible.

Mounting

The mountant:
(a) acts as a permanent bond between slide and coverslip
(b) protects cell material from air drying and shrinkage
(c) acts as a seal against oxidation and fading of the stain.

Causes of inconsistent staining

1.varying thickness of material on slide
2. type of fixative used
3. inadequate filtering of stain solutions
4. age of staining solution
5. degree of usage of staining solutions
6. use of chlorinated tap water
7. pH of water can effect nuclear staining
8. temperature of water
9. insufficient rinsing after acid
10.air drying of slides between solutions
11.improper draining of slides during staining.

CORNFLAKE ARTIFACT

This common brown artifact is said to be caused by air bubbles formed when xylol dries
before the slide is mounted. It can sometimes Be so extensive as to render the slide
unsuitable for evaluation. Remounting the slide can sometimes improve the appearance of
the smear.